Methods to Measure MDSC Immune
Suppressive Activity In Vitro and In Vivo
Samantha Solito,1,4Laura Pinton,2,4Francesco De Sanctis,3Stefano Ugel,3 Vincenzo Bronte,3Susanna Mandruzzato,1,2and Ilaria Marigo2,51Oncology and Immunology Section, Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy
2Veneto Institute of Oncology IOV- IRCCS, Padova, Italy
3Immunology Section, Department of Medicine, Verona University Hospital, Verona, Italy 4These authors contributed equally to this work
5Corresponding author: ilaria.marigo@iov.veneto.it
This unit presents methods to assess the immunosuppressive properties of immunoregulatory cells of myeloid origin, such as myeloid-derived suppressor cells (MDSCs), both in vitro and in vivo in mice, as well as in biological samples from cancer patients. These methods could be adapted to test the impact of different suppressive populations on T cell activation, proliferation, and cytotoxic activity; moreover, they could be useful to assess the influence exerted by genetic modifications, chemical inhibitors, and drugs on immune suppressive pathwaysC 2018 by John Wiley & Sons, Inc.
Keywords: adoptive cell transfer r chromium release test r flow cytometry r immunomagnetic sorting r immunosuppression r MDSC r thymidine incor-poration
How to cite this article:
Solito, S., Pinton, L., De Sanctis, F., Ugel, S., Bronte, V., Mandruzzato, S., & Marigo, I. (2019). Methods to measure of MDSC immune suppressive activity in vitro and in vivo. Current
Protocols in Immunology, 124, e61. doi: 10.1002/cpim.61
INTRODUCTION
MDSCs are immature myeloid cells with potent immune suppressive activity that are characterized by a pathological state of activation (Bronte et al., 2016; Gabrilovich et al., 2007).
Two major subsets of MDSCs have been classified based on their phenotypic and mor-phological features: monocytic (M)-MDSCs and polymorphonuclear (PMN)-MDSCs. In mice, MDSCs described in the past as CD11b+and Gr-1+ cells (Bronte et al., 2000; Dolcetti et al., 2010) are now better identified as CD11b+Ly6ChiLy6G-(M-MDSCs) and CD11b+Ly6CloLy6G+(PMN-MDSCs) (Bronte et al., 2016; De Sanctis, Bronte, & Ugel, 2016a; Dolcetti et al., 2010; Movahedi et al., 2008; Peranzoni et al., 2010).
In cancer patients, the characterization of MDSCs is more complex, although it is cur-rently accepted that three main subsets of circulating MDSCs exist, i.e., M-MDSCs and PMN-MDSCs, while a more immature population, whose counterpart in the mouse is not known, is defined as early-stage MDSCs (eMDSCs). Each of these subsets have been characterized by different combinations of myeloid markers and contain more than a single phenotype (Bronte et al., 2016; Damuzzo et al., 2015). Moreover, a recent study demonstrated that human MDSCs, under different conditions, can express additional
Current Protocols in Immunology e61, Volume 124
Solito et al.
markers such as CD38, LOX-1, and PD-L1, highlighting their great plasticity and adding additional layers of complexity to MDSC characterization and targeting (Tcyganov, Mastio, Chen, & Gabrilovich, 2018).
Basic Protocol 1 shows how to isolate MDSCs from spleens of tumor-bearing mice, and Basic Protocol 2 illustrates how to generate MDSCs from mouse bone marrow (BM) samples. MDSCs obtained using these protocols can be assessed for their ability to suppress T cell proliferation in response to antigen-specific activation (Basic Protocol 3) or following nonspecific stimulation (Basic Protocol 4). Basic Protocol 5 shows how to assess T cell cytotoxic effector function in response to antigen-specific activation, and therefore a Support Protocol describing the calculation of results is included. A method to evaluate in vivo–induced immune suppression in mice is also presented (Basic Protocol 6).
Basic Protocol 7 indicates how to isolate circulating human CD14+cells enriched in M-MDSCs. Basic Protocol 8 illustrates how to expand in vitro human bone marrow–derived MDSCs (BM-MDSC), and Basic Protocol 9 describes how to test these cells for their suppressive function on T cell proliferation.
PMN-MDSCs share the expression of the markers CD11b, CD15, (or CD66b+) and CD33 with neutrophils, but differ in their buoyant density, since PMN-MDSCs can be isolated in low-density Ficoll-gradient fraction, while neutrophils are present in the high-density fraction. Thus, the addition of a specific marker is warranted to obtain an enrichment of PMN-MDSCs. In this regard, we demonstrated that CD124 is up-regulated both in M-MDSCs and in PMN-MDSCs, although its presence only correlates with an immunosuppressive phenotype in M-MDSCs but not PMN-MDSCs of tumor-bearing patients. However, the isolation of CD124-positive versus -negative cells is technically challenging, since the staining of this antigen has a low intensity and a unimodal expression, thus making the separation of a highly enriched CD124+population difficult to achieve. More recently, lectin-type oxidized LDL receptor 1 (LOX-1) has been proposed as a marker to distinguish human neutrophils from PMN-MDSCs without the use of a density gradient (Condamine et al., 2016).
All the in vitro protocols were optimized for microcultures in order to reduce the number of cells to be used.
NOTE: All protocols using live animals must first be reviewed and approved by an
Institutional ethics committee and must be executed in accordance with governing laws, directives, and guidelines.
NOTE: Patients must provide their informed consent and Institutional ethics committees
must approve all experiments with human samples.
NOTE: All solutions and equipment coming into contact with cells must be sterile, and
proper aseptic technique must be used accordingly.
NOTE: All incubations are performed in a humidified 37°C, 5% CO2incubator unless
otherwise noted.
BASIC PROTOCOL 1
ISOLATION OF MYELOID CELL SUBSETS FOR MEASUREMENT OF IMMUNOSUPPRESSIVE ACTIVITY
This protocol is optimized to isolate MDSCs from the spleens of tumor-bearing mice, preserving their functional activity in order to be used for both in vitro and in vivo functional assays. It allows the high- purity separation of PMN-MDSCs and M-MDSCs by FACS sorting, as required for the accurate immunosuppressive assays described here Solito et al.
105 0 103 104 105 102 0 103 104 105 102 0 103 104 105 102 0 103 104 105 102
Viability gate Myeloid-gate
SSC-A Live-Dead C1 1 b D FSC-A 0 103 104 105 102 0 103 104 105 102 MDSC-gate Ly 6 C Ly6-G 0102 103 104 105 0 103 104 105 102
M-MDSC-gate (Ly6C cells)+
Ly 6 C Ly6-G 0102 103 104 105 0 103 104 105 102
PMN-MDSC-gate (Ly6G cells)+
Ly 6 C Ly6-G 0 103 104 105 102 0 103 104 105 102 Singlet gate FSC-H FSC-A SSC-A 0 103 104 102 0 103 104 105 102 FSC-A Morpho gate
Figure 1 Gating strategy to isolate mouse MDSC using flow cytometric sorting. Cell suspension from tissues (spleen, blood, or tumor) of tumor-bearing mice orin vitro–differentiated BM cells were stained with the following mix: anti-Ly6C, anti-ly6G, anti-CD11b and live–dead probe. The figure illustrates isolation of the two main MDSC subsets derived by sequential steps: a morpho gate, a viability gate, a myeloid gate, and an MDSC gate. The procedure makes it possible to isolate M-MDSCs and PMN-MDSCs with a purity of80% to 90%.
and the molecular analysis protocols. In Figure 1, the gating strategy and an example of separation are presented.
For tumor models in which low percentages of CD11b+cells accumulate in the spleen, the purity of MDSCs obtained could be lower; titration of antibodies and reagents may help in obtaining better results. Moreover, a pre-enrichment step through immunomagnetic sorting using specific CD11b microbeads could be coupled with this protocol according to the manufacturer’s instructions (see mouse and human CD11b MicroBeads, Miltenyi). This might improve the results, but it might also affect the viability and performance of the sorted cells.
This protocol could be applied to separate myeloid subsets in lymphoid organs other than spleen (i.e., bone marrow and lymph nodes) or from the tumor mass; however, an organ might present some peculiarities due to its structure (for example, it is necessary to digest the tumor to obtain a single-cell suspension) or function (for example, lymph nodes contain lower amounts of myeloid cells), which might limit either purity or viability of the recovered cell fractions.
This protocol has been used to successfully isolate different fractions of MDSCs from C26GM and 4T1 tumor-bearing BALB/c mice, and from MCA203 and MN-MCA1 tumor-bearing C57BL/6 mice. The protocol can be used to separate splenocytes of mice bearing tumors of different type and histology, provided that they induce an expansion of MDSCs. High purity is less likely to be achieved with low percentages of MDSCs. The same protocol has also been used to separate MDSC subsets obtained from bone marrow-derived MDSCs, in vitro–generated, as previously reported (Marigo et al., 2010) and as described in Protocol 2.
Previously, we published (Peranzoni et al., 2010; also see previous version of this unit; doi: 10.1002/0471142735.im1417s91) a separation protocol that described how to obtain three different subsets of myeloid cells from the spleens of tumor-bearing mice, by immunomagnetic sorting. A commercial Miltenyi kit is now available for this separation based on 1 marker expression, which allows the separation of PMN-MDSCs as Gr-1highcells (corresponding to Ly6Ghighcells) and M-MDSCs as Gr-1dimLy6G-containing different proportions of Ly-6Chigh and Ly-6Clow cells dependent on the tumor models
Ly-6G-CD11b+. This protocol is still valid in cases in which a FACS sorter is not available and when highly pure populations are not required.
Materials
Spleens from BALB/c or C57BL/6 tumor-bearing mice (mice purchased from Jackson Laboratories) or in vitro-generated MDSCs (see Basic Protocol 2) RPMI containing 3% FBS (see recipe)
Red cell ACK lysis buffer (Lonza)
Dulbecco’s phosphate-buffered saline (DPBS; Lonza BioWhittaker, cat. no. BE17-515Q), cold
Fc-receptor (FcR) blocking reagent (clone 2.4G2, ThermoFisher) Sorting buffer (see recipe) fixable viability stain
Fixable Viability Dye (ThermoFisher) Anti mouse-CD11b (ThermoFisher) Anti mouse-Ly6G (ThermoFisher) Anti mouse-Ly6C (ThermoFisher)
Fetal bovine serum (FBS), heat inactivated RPMI containing 10% FBS (see recipe) 10-mm culture dish
2-ml syringe
15- and 50-ml conical tubes Centrifuge
100-μm nylon-mesh cell strainer (BD Biosciences) MoFlo Astrios cell sorter (Beckman Coulter) LSRII flow cytometer (BD Biosciences) FlowJo 7.6.5 Software (TreeStar)
Additional reagents and equipment to harvest spleens from mice (see Reeves & Reeves, 1992) and to determine cell number using trypan blue dye exclusion (Strober, 2001)
NOTE: All antibodies need to be titrated. When working with more than one spleen and
different amounts of cells adapt reagent volume accordingly.
Process spleen(s)
1. Collect spleens of tumor-bearing mice (Reeves & Reeves, 1992) in a small volume of RPMI containing 3% FBS under sterile conditions.
Take care to clean spleen from surrounding fibrous tissue.
2. Place spleen in a 10-mm culture dish with a small volume of RPMI containing 3% FBS and gently disaggregate using the plunger of a 2-ml syringe.
If more than one spleen has to be collected to perform more tests, spleens can be stored in a small volume of RPMI containing 3% FBS in a 50-ml conical tube on ice.
3. Add 5 ml of RPMI containing 3% FBS to cells and, pipetting gently, collect the cells in a 50-ml conical tube. Repeat steps 2 and 3 until the complete disaggregation of the spleen has been achieved.
4. Centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant.
The supernatant may be discarded by decantation or aspiration.
5. Lyse red blood cells by adding a volume of 5 ml (for each spleen) of red cell ACK lysis buffer to the pelleted cells, and incubate at room temperature for 4 min. 6. Add a volume of RPMI containing 3% FBS equal to at least five times the volume
of lysis buffer added, and mix by gently pipetting up and down. Solito et al.
7. Centrifuge the suspension 6 min at 300× g, 4°C, and discard the supernatant. 8. Resuspend cell pellet in 10 ml/spleen of RPMI containing 3% FBS, filter through a
cell strainer (or a single sheet of nylon mesh) placed on the top of a 50-ml tube, and collect the suspension.
Filtration can be helped by pre-hydrating the nylon mesh with a small volume of medium.
9. Properly dilute an aliquot of the single-cell suspension with trypan blue solution (Strober, 2001) and estimate the number of viable cells, avoiding red blood cell counts.
Trypan blue colors dead cells a faint blue, and these cells should not be taken into account. Red blood cells can be recognized as small cells with a round shape and a neat, thick perimeter.
Separation of PMN-MDSCs and M-MDSCs from splenocytes
An example of gating strategy for the sorting and results analyzed with FlowJo 7.6.5 Software is shown in Figure 1.
10. Transfer 1× 108viable cells obtained from the spleens of tumor-bearing mice to a new 15-ml conical tube, and then add 8 to 10 ml of DPBS to wash the cells.
In the case of spleens coming from mice with completely unknown tumors, the proportion of myeloid cells should be evaluated before staining a large sample.
11. Centrifuge the suspension 6 min at 300× g, 4°C, and discard the supernatant. 12. Add 10 ml of sorting buffer and wash cells by gently pipetting up and down. 13. Centrifuge the suspension 6 min at 300× g, 4°C, and discard the supernatant.
These fractions can be used to assess their immunosuppressive activity either in vitro and in vivo.
14. Incubate samples with 10μl Fc-receptor blocking reagent in 50 μl of sorting buffer at room temperature for 10 min.
15. Add the mixture of antibody, composed of Fixable Viability Dye (5μl), anti-CD11b (10 μl), anti Ly6G (10 μl), and anti Ly6C (10 μl), to the tubes and incubate at 4°C for 20 min. Adjust the volume of staining mix to 100 μl with the sorting buffer. Specifically, for FACS analysis, cells are stained in 15-ml polypropylene tubes previously coated for at least 1 hr with heat-inactivated fetal bovine serum. 16. Wash twice with sorting buffer. Centrifuge the suspension 6 min at 300× g, 4°C,
and discard the supernatant.
17. After labeling, resuspend samples at the concentration of 30 × 106 cells/ml of
sorting buffer and proceed with the FACS separation as outlined in Figure 1. 18. Filter MDSCs through a 100-µm cell strainer and isolate through MoFlo Astrios.
Sorting should be performed with a 100-μm nozzle, setting the pressure, voltage, and cell rate as appropriate for the sorter.
19. Collect sorted M-MDSCs and PMN-MDSCs in two different 15-ml polypropylene tubes previously coated for at least 1 hr with heat-inactivated fetal bovine serum. 20. After the separation, wash and resuspend M-MDSCs and PMN-MDSCs in 10 ml
of RPMI/10% FBS. Take an aliquot of the single-cell suspension, dilute it properly with trypan blue solution, and estimate the number of viable cells (Strober, 2001).
NOTE: All the procedures, including the sorting, are performed at 4°C, to avoid cell loss and adherence to the plastic.
These fractions can be stained to assess the presence and distribution of markers that were used to characterize mouse MDSCs (Table 1).
BASIC PROTOCOL 2
MOUSE BM-MDSC GENERATION
This protocol is optimized to obtain bone marrow (BM)–derived MDSCs from mice as previously described (Marigo et al., 2010). BM cells are cultured for 4 days in the presence of the recombinant mouse cytokines GM-CSF and IL-6. The final cultures will contain proportions of PMN-MDSCs, M-MDSCs, and macrophages.
Materials
C57BL/6 mice (The Jackson Laboratory) RPMI containing 3% FBS (see recipe) Red cell ACK lysis buffer (Lonza) RPMI containing 10% FBS (see recipe) Premium Grade IL-6 (Miltenyi Biotec) Premium Grade GM-CSF (Miltenyi Biotec)
DPBS without Ca or Mg (Lonza BioWhittaker, cat. no. BE17-515Q) containing 2 mM EDTA
Scissors and pliers
2-ml syringe with 26-G needle 15- and 50-ml conical tubes Centrifuge
100-μm nylon-mesh cell strainer (BD Biosciences) 6-well culture dishes
Additional reagents and equipment to harvest bone marrow from mice (Reeves & Reeves, 1992) and to determine cell number using trypan blue dye exclusion (Strober, 2001)
Process bone marrow (BM)
1. Remove tibias and femurs from mice, and remove the muscle from the bones with scissors.
Take care to avoid hair contamination
2. Cut the extremity of the bones with pliers and scissors under sterile conditions. 3. Flush the medium inside the bones with a small volume of RPMI containing 3%
FBS, gently injected with a 2-ml syringe with a 26-G needle. Collect flushed-out material in 50-ml conical tubes containing 5 ml of RPMI containing 3% FBS
BM-MDSC generation
4. Centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant.
The supernatant may be discarded by decantation or aspiration.
5. For two legs, lyse red blood cells by adding a volume of 5 ml of red blood cell lysis buffer to the pelleted cells, and incubate at room temperature for 4 min.
6. Add a volume of RPMI/3% FBS equal to at least five times the volume of lysis buffer added, and mix by gently pipetting up and down.
7. Centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant. Solito et al.
Ta b le 1 Common mAbs Used fo r C har acter ization of Surf ace Molecules in Mouse M DSCs Epitope Clone Antibody type Compan y E xpression CD11b M1/70 Rat IgG2b, κ ThermoFisher + Gr-1 RB6-85C Rat IgG2b, κ Biole gend + L y6G 1A8 Rat IgG2a, κ ThermoFisher ± L y6C Mk1.4 R at IgM, κ ThermoFisher ± CD115 AFS98 Rat IgG2a, κ ThermoFisher Lo w/ − CD16/CD32 2.4G2 Rat IgG2b, κ BD Pharmingen + CD124 (IL-4R α ) mIL4R-M1 R at IgG2a, κ BD Pharmingen ± CD40 3/23 Rat IgG2a, κ BD Pharmingen − CD80 16-10A1 Armenian Hamster IgG2, κ BD Pharmingen + CD86 GL1 Rat IgG2a, κ BD Pharmingen − MHC-I (H-2Kd) SF1-1.1 M ouse IgG2a, κ BD Pharmingen + MHC-I (H-2Kb) AF6-88.5 M ouse IgG2a, κ BD Pharmingen + MHC-II 2G9 R at IgG2a, κ BD Pharmingen − CD31 390 Rat IgG2a, κ Biole gend + /Lo w /− F4/80 A3-1 Rat IgG2b Serotec Lo w/ − CD2 R M2-5 Rat IgG2a, λ BD Pharmingen Lo w/ − CD71 RI7217 Rat IgG2a, κ Biole gend Lo w/ − CD11c N418/HL3 Armenian Hamster IgG/Armenian Hamster IgG1, λ 2 Biole gend/BD Pharmingen Lo w/ − ER-MP58 ER-MP58 Rat IgM Serotec + Solito et al.
8. Resuspend the cells obtained from two legs in 5 ml of RPMI containing 10% FBS and filter the suspension through a cell strainer.
The volume can be scaled to meet researchers’ needs; extra medium volume can also be added to rinse the cell strainer, minimizing cell loss.
9. Take an aliquot of the single-cell suspension, dilute it properly with trypan blue solution, and estimate the number of viable cells (Strober, 2001).
10. Adjust the concentration of the BM suspension to 1.5 × 106 cells/ml using RPMI/10% FBS.
11. Plate 1 ml (1.5× 106 cells) of BM cells in each well of a 6-well plate, add 2 ml of RPMI/10% FBS in the presence of cytokines GM-CSF and IL-6, both at a final concentration of 40 ng/ml (per well), and place the culture in a 37°C, 5% CO2
incubator for 4 days.
12. At day 4 of culture, to test the immunosuppressive properties of the cells, gently harvest BM-MDSCs with a Pasteur pipette. Wash dishes with DPBS containing 2 mM EDTA to detach and collect the remaining cells.
BASIC PROTOCOL 3
MEASURING MYELOID-INDUCED SUPPRESSION OF T CELL ANTIGEN-INDUCED PROLIFERATION IN VITRO BY CELL TRACE DILUTION
Immune suppression exerted by myeloid cells to the detriment of activated T cells can be measured in terms of inhibition of proliferation by evaluating dye dilution to trace multiple generations of proliferating lymphocytes.
Materials
Immunosuppressive cells, e.g., MDSCs (Basic Protocol 1 or 2) RPMI containing 10% FBS (see recipe)
CD45.1 congenic mice; purchased from Jackson Laboratories, under the name B6.SJL-PtrcaPepcb/BoyJ.
Transgenic OT-1 mice on a C57BL/6 background bearing aαβ T cell receptor (TCR) that recognizes the Kb-restricted OVA257-264peptide; purchased from
Jackson Laboratories, under the name C57BL/6-Tg (TcraTcrb)1100Mjb/J) Red cell ACK lysis buffer (Lonza)
RPMI containing 3% FBS (see recipe)
Dulbecco’s phosphate-buffered saline (DPBS without Ca and Mg; Lonza Biowhittaker, cat. no. BE17-515Q)
CellTraceTMViolet Cell Proliferation Kit (Molecular Probes) Fetal bovine serum (FBS)
10 mg/ml OVA257-264 peptide stock solution (available lyophilized from JPT
Peptide Technologies) Staining buffer (see recipe)
Fc-receptor (FcR) blocking reagent (clone 2.4G2, ThermoFisher)
Anti-CD8 (i.e., PeCy5-anti CD8a, clone 53.6.7, cat. # 15-0081-81, ThermoFisher) Anti-CD45.2 conjugated to a brilliant fluorochrome (i.e., PE-anti CD45.2, clone
104, cat. # 12-0454-81, ThermoFisher) 96-well or 384-well flat-bottom plates 10-mm culture dish
2-ml syringe 50-ml conical tube Refrigerated centrifuge
100-μm nylon-mesh cell strainer (BD Biosciences) Solito et al.
LSRII flow cytometer (BD Biosciences) FlowJo 7.6.5 Software (TreeStar) 4-ml round-bottom tubes
TruCount Tubes (Becton Dickinson)
Additional reagents and equipment to prepare immunosuppressive cells (Basic Protocol 1 and 2), harvest spleens from mice (Reeves & Reeves, 1992), and count viable cells by trypan blue exclusion (Strober, 2001)
Day 0: Suppressive cell plating
1. Prepare MDSCs, according to Basic Protocol 1 or 2.
2. Wash cells twice in RPMI medium containing 10% FBS and resuspend in a suitable volume of RPMI containing 10% FBS.
3. Dilute an aliquot of suspension in trypan blue solution and determine viable cell concentration (Strober, 2001).
4. Adjust cell concentration appropriately with RPMI/10% FBS.
The concentration will depend upon the design of the experiment. A good range of MDSCs is 1.5% to 24% of the effector culture cellularity. For the 96-well plate, if 0.6× 106effector cells are used, MDSC concentration can be adjusted to 1.44× 106/ml,
so that 0.144× 106 cells will be plated in 100μl (24% of the effector cells). For the
384-well plate, MDSC concentration can be adjusted to 0,6× 106/ml, so that 24,000 cells
will be plated in 40μl (24% of the effector cells).
For example, in the 96-well plate, if the experimental setup requires 3% of suppressive cells in the coculture and effector splenocytes are 0.6 × 106 cells/well, 0.018 × 106
suppressive cells will be plated per well. Adjust suppressive cell concentration to 0.18× 106cells/ml and plate 100μl/well.
5. Plate myeloid cells in triplicate in a 96-well flat-bottom microplate in a volume of 100μl (or 40 μl for 384-well plate) of RPMI/10% FBS, and place the microplate in a 37°C, 5% CO2incubator for at least 30 min.
This assay requires a careful titration of the suppressive cells. The number of suppressor cells to add to each well will depend on the experimental plan. A good starting point could be to plate suppressor cells at 24% of the total cells in control cultures and serially dilute them by a factor of 2 until they represent1.5% of total cells. When organizing the distribution of samples, avoid using the outer wells, because these wells are more susceptible to evaporation. Outer wells can be filled with RPMI containing 3% FBS. Remember to fill at least three wells with RPMI/10% FBS without myeloid suppressor cells, to use as control cultures.
Plating splenocytes
6. Separately collect spleens from both CD45.1 and OT-1 mice (Reeves & Reeves, 1992) in a small volume of RPMI/3% FBS under sterile conditions.
Splenocytes from OT-1 mice need to be diluted with CD45.1 splenocytes to obtain a concentration of OVA-specific CD8+T lymphocytes, which is 1% of total cultured cells (usually about 1:10 dilution is sufficient). Consequently, one spleen from OT-1 mice is sufficient for several microtiter plates. The percentage of specific OVA CD8+T lympho-cytes in total splenolympho-cytes can be determined before their use by cytometry staining with anti-CD8 and anti-Vα2 Vβ5.1/5.2 mAbs to identify the specific T cell receptor (TCR).
7. Place spleens in a 10-mm culture dish with a small volume of RPMI/3% FBS, and gently disaggregate using the plunger of a 2-ml syringe.
8. Add 5 ml of RPMI/3% FBS to cells and collect the cells in a 50-ml conical tube by gently pipetting up and down. Repeat steps 7 and 8 until the spleen is completely
9. Centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant. 10. Lyse red blood cells by adding about 5 ml of red cell lysis buffer for spleen, and
incubate at room temperature for 4 min.
11. Add a quantity of RPMI/3% FBS equal to at least five volumes the quantity of lysis buffer applied, and mix by gently pipetting up and down.
It is very important to obtain a red blood cell–free suspension (see explanation in Commentary).
12. Centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant. 13. Resuspend cells in 1 ml of RPMI/10% FBS, and put on ice.
14. Wash both CD45.1 and OT-1 cells by adding 10 ml of RPMI/10% FBS. 15. Centrifuge the suspensions 6 min at 300×g, 4°C, and discard the supernatant. 16. Resuspend the cells in 2 ml/spleen of RPMI/10% FBS, and filter the suspension
through a cell strainer.
17. Dilute an aliquot of suspension in trypan blue solution, and determine viable cell concentration (Strober, 2001).
18. Keep CD45.1 splenocytes on ice and wash OT-1 cells twice by adding 10 ml of ice-cold DPBS (4°C) and discarding the supernatant.
19. Adjust OT-1 splenocyte concentration to 20× 106cells/ml concentration with room
temperature DPBS and add an equal volume of DPBS containing CellTraceTM at the concentration of 5μM (the final concentration of the suspension will be equal to half the concentration). Keep the suspension 5 min at 37°C in incubator and mix every minute.
20. Block the staining by adding an equal volume of FBS and keep at room temper-ature for 1 min. Centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant.
21. Wash cells twice in DPBS with 10% FBS and resuspend in 5 ml of RPMI. 22. Check the staining with CellTraceTMby flow cytometry.
When evaluating CellTrace incorporation, a control for autofluorescence value of un-stained splenocytes should be included, to evaluate whether the difference in emission signals among unstained splenocytes and CellTrace+splenocytes is enough to be able to quantify extensive proliferation, which will bring a strong dilution of CellTrace signal. A successful staining is achieved when the histogram has a single, narrow peak.
23. Adjust the concentration of both the CD45.1 and the OT-1 splenocyte suspensions to 12× 106cells/ml for 96-well plate (or at 5× 106cells/ml for 384 wells/plate) in RPMI/10%.
24. Mix CD45.1+ and CellTrace+ OT-1 cells in appropriate proportions to obtain a concentration of OVA-specific CD8+T lymphocytes that is 1% of the total culture, and add OVA257-264peptide to a final concentration of 1μg/ml.
CD8+OVA-TCR+percentage in OT-1 mice can be determined by FACS analysis using a simple staining with anti-CD8α and anti-anti-Vα2 Vβ5.1/5.antibodies. CD8+OVA-TCR+ double-positive cells are usually about 10% of total splenocytes in these transgenic mice, while the same cell population is almost negligible in wild-type C57BL/6 or congenic CD45.1 mice. In order to obtain a cell suspension with 1% CD8+OVA-TCR+ cells, 1 part of OVA splenocytes (with 10% CD8+OVA-TCR+cells) can be added to 9 parts of CD45.1+splenocytes (with0% CD8+OVA-TCR+cells).
25. Immediately plate 50μl per well of cell suspension onto the previous 96-well plate containing the immunosuppressive myeloid cells (step 5), or 20 μl per well if a 384-well plate was used.
26. Add 50μl to 96-well plate (or 20 μl for 384-well plate) of RPMI/10% FBS contain-ing OVA257-264peptide at a final concentration in each well of 1μg/ml. Incubate for
3 days in the 37°C, 5% CO2incubator.
Day 3: Culture harvesting
27. Pool triplicate cultures to new 4-ml round-bottom tubes, wash samples once with staining buffer, centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant.
28. Block nonspecific binding with 50μl FcR blocking reagent for 10 min at 4°C. 29. Stain cells with 50 μl of a mix composed of 0.5 μl of anti-CD8 and 0.1 μl of
anti-CD45.2 antibodies in 49μl of staining buffer, for 20 min at 4°C.
Remember to include appropriate single-staining controls.
30. Wash samples once with staining buffer, centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant.
31. Resuspend samples in 200 μl DPBS, transfer the mix in TruCount tubes, briefly vortex and then proceed with flow cytometric acquisition and analysis.
Flow cytometric analysis
32. Perform flow cytometric acquisition and analysis.
To detect proliferating CD8+T cells, gate cells first by their morphology, and then collect a sufficient number of events in the CD8+/CD45.2+/CellTrace+gate.
After the acquisition, proceed with data analysis. Gate cells by morphology and iden-tify CD8+/CD45.2+/CellTrace+ population. Inside this gate, set a baseline gate on the histograms of CellTrace signal of unstimulated splenocyte controls, and copy this gate to the stimulated CD8 cells cultured in the presence or absence of MDSCs, in order to determine the percentage of proliferating T lymphocytes. With FlowJo, it is the possible to model proliferation data; FlowJo presents a graphical display as well as information about each cell generation in the subset. The proliferation platform also provides infor-mation about the fraction of cells from the original population that have divided and the number of times these cells have divided. In addition, the FlowJo Proliferation Platform draws gates that separate each cell generation.
Results obtained can be quantitative or qualitative. See Critical Parameters and Trou-bleshooting.
BASIC PROTOCOL 4
MEASURING MYELOID-INDUCED SUPPRESSION OF T CELL
PROLIFERATION IN VITRO BY ANTI-CD3/ANTI-CD28 STIMULATION AND
EVALUATION OF [3H]THYMIDINE ([3H]TdR) INCORPORATION
Proliferation of T cells can be induced by antigen-independent stimulation, and MDSCs can exert immune suppressive functions on these cells. The proliferative arrest of actively dividing cells can also be measured as a function of [3H]TdR incorporation by T cells into their DNA. The method is extremely simple and provides a quantitative and rela-tively rapid analysis, which can be easily applied to complex experiments as large-scale screenings of drugs and treatments.
Materials
Dulbecco’s phosphate-buffered saline (DPBS; Lonza BioWhittaker, cat. no. BE17-515Q)
Anti-CD3 (2C11, ATCC) Anti-CD28 (clone 37.5, ATCC)
Immunosuppressive (myeloid) cells, e.g., MDSCs (Basic Protocol 1 or 2) RPMI containing 10% FBS (see recipe)
C57BL/6 mice purchased from Jackson Laboratories RPMI containing 3% FBS (see recipe)
Red cell ACK lysis buffer (Lonza) [3H]TdR (PerkinElmer)
Serum-free RPMI (e.g., Invitrogen)
Serum-free RPMI medium (e.g., Invitrogen) 96% ethanol
MicroScint-20 scintillation fluid (PerkinElmer) 96-well flat-bottom microtiter plate
10-mm culture dish2-ml syringe 50-ml conical tubes (BD Falcon) Refrigerated centrifuge
100-μm nylon-mesh cell strainer (BD Biosciences) Unifilter-96 GF/C plate (PerkinElmer) with plate sticker 96-well U-bottom microtiter plate (PerkinElmer) Plate harvester: FilterMate 196 (Packard)
TopSeal-A 96-well microtiter plate adhesive sealers (PerkinElmer) Scintillation counting device (TopCount, PerkinElmer)
Computer running spreadsheet program, e.g., Microsoft Excel
Additional reagents and equipment to prepare immunosuppressive cells (Basic Protocol 1 or 2), harvest spleens from mice (Reeves & Reeves, 1992), and count viable cells by trypan blue exclusion (Strober, 2001)
Day 0: Culture plate coating
1. Prepare sufficient coating buffer: DPBS containing anti-CD3 (3μg/ml final concen-tration) and anti-CD28 (2μg/ml final concentration).
Antibody concentration may be optimized on the basis of stock and supplier; a suitable volume would be 11 ml of antibody solution per plate.
2. Fill 96-well flat-bottom microtiter plates with 100μl/well anti-CD3 and anti-CD28-containing DPBS with a multichannel pipettor; also fill an equal number of wells with 100μl/well of DPBS without antibodies, for nonspecific proliferation measurement.
Also remember to fill extra wells for appropriate controls based on the experimental set up, e.g., suppressive cells only.
3. Incubate the plate overnight at 4°C.
Day 1: Cell plating
Plate immunosuppressive cells
4. Prepare myeloid (immunosuppressive) cells, e.g., MDSCs, according to Basic Pro-tocol 1 or 2 or other strategies of enrichment.
5. Wash cells twice in RPMI containing 10% FBS and resuspend them in a suitable volume of RPMI/10% FBS.
The supernatant should be aspirated from the top; be careful to remove as much super-natant as possible without disturbing the cell pellet.
6. Resuspend cells in a small volume of RPMI/10% FBS. Dilute an aliquot of sus-pension in trypan blue solution and determine viable cell concentration (Strober, 2001).
7. Adjust cell concentration appropriately with RPMI/10% FBS.
The concentration depends upon the design of the experiment. A good range of MDSCs is 24% to 1.5% of the effector culture cellularity. If 0.6× 106 effector cells are used,
MDSC concentration can be adjusted to 1.44× 106/ml, so that 0.144× 106cells will be
plated in 100μl (24% of the effector cells).
8. Working under sterile conditions, empty 96-well microtiter plate (from step 3) by inverting it with a rapid movement.
Work under a laminar flow hood; a plastic basin wrapped with paper towels can be used to discard the coating buffer.
9. Fill every well with 200μl of DPBS using a multichannel pipettor and empty the plate as in step 8. Repeat at least three times to wash extensively.
Fill the plate from the top of the well to avoid scratching the surface of the well, which might disturb the antibody coating.
10. Immediately plate suppressive cells in triplicate for specific and background prolif-eration in 100μl of RPMI/10% FBS, and place the microtiter plate in a 37°C, 5% CO2incubator.
When organizing the distribution of samples, avoid using the outer wells, because these wells are more susceptible to evaporation. Outer wells can be filled with sterile medium. Remember to fill at least six wells with RPMI/10% FBS without suppressive cells; these wells will be used as control cultures for the determination of specific and nonspe-cific proliferation. Multiply these control wells for different treatments according to the experimental setup, e.g., different inhibitors and drugs to be tested.
Plate splenocytes
11. Collect spleens from C57BL/6 mice (Reeves & Reeves, 1992) in a small volume of RPMI containing 3% FBS under sterile conditions.
Usually one spleen from an 8-week old-mouse is sufficient for one plate.
12. Place spleens in a 10-mm culture dish with a small volume of RPMI/3% FBS and gently disaggregate them using the plunger of a 2-ml syringe.
13. Add 5 ml of RPMI/3% FBS to cells and collect the cells in a 50-ml conical tube by gently pipetting. Repeat steps 12 and 13 until complete disaggregation of the spleen is achieved.
14. Centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant. 15. Lyse red blood cells by adding a volume of red cell ACK lysis buffer equal to the
volume of the pelleted cells, and incubate at room temperature for 4 min.
16. Add a volume of RPMI/3% FBS equal to at least five times the volume of lysis buffer added, and mix by gently pipetting up and down.
17. Centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant. 18. Wash cells with 10 ml of RPMI/10% FBS.
19. Centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant. 20. Resuspend the cells in 2 ml of RPMI/10% FBS for each spleen and filter the
The volume can be scaled to meet researchers’ needs; extra medium volume can also be added to rinse the cell strainer, preventing cell loss.
21. Dilute an aliquot of the suspension in trypan blue solution and determine viable cell concentration (Strober, 2001).
22. Adjust the concentration of the splenocyte suspension to 6 × 106 cells/ml with
RPMI/10% FBS.
23. Immediately plate 100μl of cell suspension onto the previously coated microtiter plate and incubate 3 days in the 37°C, 5% CO2incubator.
Pay attention to the cell distribution schema. Splenocytes will proliferate upon anti-CD3/anti-CD28 stimulation, and their proliferation will be inhibited by immunosup-pressive cells; therefore, splenocytes should be distributed in previously anti-CD3/anti-CD28-coated wells, either with or without immunosuppressive cells, as test and reference wells, respectively. The proliferative behavior of MDSCs alone under these conditions should also be tested; therefore, MDSCs will be cultured with 100μl of medium instead of effector cells.
[3H]TdR should be added the third day after culture setup. It would be useful to check the
cell culture daily in order to avoid medium exhaustion by excessive proliferation of cells (signaled by medium turning yellow) or initial cell death (granular, small cells instead of cell clumps).
Day 4
24. Add 1 μCi of [3H]TdR in 25 μl of serum-free RPMI to each well and incubate 18 hr in the 37°C, 5% CO2incubator.
The concentration of the [3H]TdR solution added to each well will thus be 0.04μCi/μl.
Day 5
Culture harvesting
25. Load a Unifilter-96 GF/C plate and an empty clean 96-well U-bottom plate onto the plate harvester and prepare Unifilter-96 GF/C plate by washing it with distilled water.
A wet Unifilter-96 GF/C is essential to obtain consistent results.
26. Load the culture plate onto harvester and aspirate culture medium, collecting it in the “hot” radioactive waste tank.
27. Wash the plate five times with water and aspirate, collecting the supernatant in the “hot” radioactive waste tank. Repeat another five times using aspiration, collecting the supernatant in the “cold” radioactive waste tank.
28. Remove the culture plate from the harvester and replace it with a proper vessel filled with 96% ethanol.
In this case, a proper vessel is a container, similar in dimensions to a 96-well plate, that can be accommodated properly in the harvester; it could be, for example, a plate lid or a pipet-tip box lid with the same length and width as a 96-well plate, deep enough to contain 2 to 5 ml of alcohol.
29. Completely aspirate ethanol, collecting it in the “cold” radioactive waste tank. 30. Open the harvester and let the Unifilter-96 GF/C plate dry by continuous aspiration.
Plate dryness is essential to obtain consistent results, since the scintillation cocktail can only be mixed with a very small amount of water and still perform correctly.
Preparation of Unifilter-96 GF/C plate for reading
31. Check plate dryness.
The Unifilter-96 GF/C plate can be observed against a light source; wells that are not properly dried will appear translucent. Wait until the plates are completely dry.
32. Apply a plate sticker on the back of the Unifilter-96 GF/C plate.
Take care to properly position the sticker. Inappropriate positioning will result in leakage of scintillation liquid.
33. Fill each well with 25μl Microscint-20 with a multichannel pipettor.
Take care not to spill the liquid outside of the well rim, since this could hinder plate sealing.
34. Seal the plate with TopSeal-A plate sealer. 35. Keep the plate in the dark for at least 30 min.
This step of incubation in the dark is necessary in order to quench the scintillation cocktail.
36. Read the plate in a TopCount scintillation counting device for 1 min/well. 37. Interpret results.
Proliferation is represented in counts per minute (cpm), which is proportional to the [3H]TdR that cells have incorporated in their DNA during the S phase of proliferation. In a spreadsheet program like Excel, calculate average cpm from triplicate cultures and subtract nonspecific proliferation. Also check for MDSC proliferation, which should be minimal.
BASIC PROTOCOL 5
MEASURING MYELOID CELL–INDUCED SUPPRESSION OF T CELL CYTOTOXIC ACTIVITY IN VITRO: INHIBITION OF ANTIGEN-INDUCED CYTOTOXIC ACTIVITY OF T CELLS IN MICROCULTURES
Immune suppression exerted by myeloid cells to the detriment of activated T cells could also be measured in ways that can complement simply looking at the process of T cell expansion. Assessing the cytotoxic activity of CD8+T cells, elicited by antigen-specific stimulation in vitro, will allow one to simultaneously assess both T cell abundance and functional effector state.
Materials
Immunosuppressive cells, e.g., MDSCs (Basic Protocol 1 and 2) RPMI containing 10% FBS (see recipe)
10 mg/ml OVA257-264peptide stock solution (available lyophilized from JPT
Peptide Technologies)
EL4 cell line (ATCC TIB-39TM) maintained in culture in 75-cm2culture flasks:
prepare a sufficient number of flasks containing EL4 cells, which need to be subconfluent on day 5
Dulbecco’s phosphate buffered saline (DPBS without Ca or Mg; Lonza BioWhittaker, cat. no. BE17-515Q)
Fetal bovine serum (FBS) 1 mCi/ml Na51CrO
4(PerkinElmer)
RPMI containing 5% FBS (see recipe) Sodium dodecyl sulfate (SDS)
96-well flat bottom microplates
Refrigerated centrifuge 12-ml round-bottom tubes
Microtiter plate carrier for centrifuge LumaPlate (PerkinElmer)
Scintillation counting device (TopCount, PerkinElmer)
Additional reagents and equipment to prepare immunosuppressive cells (Basic Protocol 1 or 2), to prepare splenocytes (Basic Protocol 3), and count viable cells by trypan blue exclusion (Strober, 2001)
Day 0: Suppressive cell plating
Prepare myeloid (immunosuppressive) cells, e.g., MDSCs, according to Basic Protocol 1 or 2 and plate them by following the procedures (1 to 5) reported in Basic Protocol 3, considering a 96-well flat-bottom microplate only.
Plating effector splenocytes
Prepare splenocytes according to Basic Protocol 3 from step from 6 to 18. Continue with the steps below.
1. Adjust the concentration of both the CD45.1 and the OT-1 splenocyte suspensions (prepared as described in Basic Protocol 3) to 6× 106cells/ml for a 96-well plate in
RPMI/10% FBS.
2. Mix CD45.1 and OT-1 splenocytes in appropriate proportions to obtain a concen-tration of OVA-specific CD8+T lymphocytes, which is 1% of the total culture, and add OVA257-264peptide for a final concentration of 1μg/ml.
See Basic Protocol 3, step 24, for details.
3. Immediately plate 100 μl of splenocyte suspension onto the previous microplate containing the immunosuppressive myeloid cells, and incubate for 5 days at 37°C, in the 5% CO2incubator.
Day 5: Target cell preparation
4. Collect EL4 in suspension cells from 75-cm2flasks in a 50-ml conical tube. 5. Centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant. 6. Resuspend the cells in 5 ml of 10% FBS in DPBS.
7. Dilute an aliquot of suspension in trypan blue solution and determine viable cell concentration (Strober, 2001).
8. Aliquot 6× 106cells to two new 12-ml round-bottom tubes. Bring volume to 10 ml with 10% FBS in DPBS.
3× 106target cells are sufficient for at least 15 test plates. The quantity can be scaled
when necessary; nevertheless, if more than 3× 106cells are needed, prepare different
aliquots for the next steps.
9. Centrifuge the suspension 6 min at 300× g, 4°C, and carefully discard the super-natant.
In this step, it is essential to eliminate as much supernatant as possible, in order to improve51Cr uptake by target cells.
10. Add 10μl of undiluted FBS to both pellets.
If you decide to load a smaller number of cells, simply scale down FBS to obtain a 10% final concentration.
11. Add 3μl of OVA257-264peptide solution (10 mg/ml) to one of the two pellets (which
will be the pulsed sample).
One pellet will not receive the peptide and will be used as control for nonspecific lysis. An EG7 cell line stably transfected to express the antigen OVA257-264is also available.
These cells can also be used in the test to evaluate specific lysis.
12. Add 100μl of 1 mCi/ml Na51CrO4to each tube and resuspend by gentle flicking.
Avoid the formation of bubbles.
13. Incubate 1 hr at 37°C, flicking the tube every 15 min. After 1 hr, wash the cells twice with RPMI containing 5% FBS, each time centrifuging the cells 10 min at 300 ×g, room temperature, removing the supernatant, and then resuspending the cells in 6 ml of RPMI/5% FBS. Determine viable cell concentration (Strober, 2001) and adjust to 0.02× 106cells/ml with RPMI/5% FBS.
During the 1-hr target incubation, proceed with the rest of the protocol (step 14). If test plates (step 14) are not ready when the target incubation ends, return washed target cells to the incubator; it is extremely important to count and adjust target-cell concentration just before plating them, to improve reproducibility.
Test plate setup
14. Fill new 96-well plate (test plate) with 100μl/well of RPMI containing 5% FBS. Resuspend cells in culture plate by gently pipetting up and down several times with a multichannel pipettor, then transfer 50μl of culture to the first row of the test plate; pipet up and down five times to mix, and then transfer 50μl of cells to the subsequent rows, thus obtaining an 8-point, 1:3 serial dilution (Fig. 2).
Every well of the culture plate has to be diluted eight times; consequently, four different culture conditions in triplicate could be arranged per each test plate. See Figure 2 as an example. Remember that specific and nonspecific lysis need to be determined in separate plates, because the same culture will be tested against peptide-pulsed and unpulsed targets: therefore, every test plate has to be prepared in duplicate. Also, remember to fill at least six wells in a separate plate (three for peptide-pulsed target cells and three for unpulsed target cells) with RPMI containing 5% FBS and 1% SDS and six wells with RPMI/5% FBS without any addition of culture cells, to evaluate maximum and spontaneous51Cr release of target cells, respectively.
15. Fill plates with 100μl of either OVA257-264peptide-pulsed or unpulsed EL4 cells
(see steps 12 to 14).
Remember to add peptide-pulsed and unpulsed target cells to wells set for maximum and spontaneous release evaluation.
16. Using a centrifuge with a microtiter plate carrier, spin down cells for 10 sec at 300 ×g, at room temperature, to bring the cells to the bottoms of the wells.
17. Incubate the plate 5 hr at 37°C in the, 5% CO2incubator.
Reading test plates
18. With a multichannel pipettor, transfer 30μl of supernatant from each well of the in-cubated test plates to a PerkinElmer LumaPlate and let medium evaporate overnight.
The same row of tips can be used to transfer a full plate, starting from the bottom (more diluted row) of the test plate to the top.
A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 Test 1 Test 2 Test 3
Control culture Suppressive cell
serial dilution
Culture Plate
Border wells (to be avoided) Culture wells
50 µl A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 50 µl 1:3 serial dilution of culture/ effector cells
Test Plate A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 Count Plate
30 µl (for each well)
5
days
5
hours
18
hour
drying
A
B
C
Figure 2 Sample protocol sheets for immunosuppressive assays. Design (A) allows measure-ment of the suppressive activity of 5-fold serial dilution of myeloid cells/plate. Designs (B) and (C) allow one to test the effector function of two lanes of cultures in plate A, with 8-fold serial dilutions.
19. Read LumaPlates in a TopCount scintillation counting device for 1 min/well.
Figure 2 shows a schematic representation of sample plating in the different steps of this protocol.
Interpretation of results and calculation of lytic units
51Cr release due to lysis of target cells, measured by the TopCount device (see Basic
Protocols 4 and 5), is expressed in counts per minute (cpm). TopCount makes it possible to set up reading protocols that can directly transform cpm values to percentage lysis, Solito et al.
Table 2 Common mAbs Used for Characterization of Human CD11blow/– BM-MDSCs and PB M-MDSCs
Epitope Clone Antibody type Company Expressiona
CD3 UUCHT1 Mouse IgG1,κ Beckman Coulter −
CD4 SK3 Mouse IgG1,κ BD Biosciences −
CD8 SK1 Mouse IgG1,κ BD Biosciences −
CD19 HIB19 Mouse IgG1,κ BD Biosciences −
CD56 NCAM16.2 Mouse BALB/c IgG2b,κ BD Biosciences −
CD11b Bear1 Mouse IgG1 Beckman Coulter Low/−
CD16 REA423 Recombinant human IgG1 Miltenyi Biotec −
CD124 25463 Mouse IgG2a R&D Systems +
CD14 M5E2 Mouse IgG2a,κ BD Biosciences +
HLA-DR L243 Mouse BALB/c IgG2a,κ BD Bioscience Low/−a
aCD11b and CD16 expressions refers to BM-MDSC, while CD124, CD14, and HLA-DR expressions refer to PB
M-MDSC.
and also calculate the average of triplicate measurements; calculations can otherwise be performed with a software like Excel applying the following formula:
% lysis=cpmexperimental− cpmspontaneous cpmmaximum− cpmspontaneous × 100
Results can be displayed in a line plot with the x axis representing culture dilution and the
y axis representing percentage of lysis. Nonspecific lysis, if any was detected, could be
subtracted from specific lysis point by point, or otherwise could be separately displayed. Another way to represent the results, which is more straightforward and also makes it possible to average more experiments, is the calculation of Lytic Units (L.U.). This involves determination of the culture dilution necessary to obtain a given lysis percentage, usually 30% (L.U.30) or 50% (L.U.50). Such determinations could be achieved using any software allowing for nonlinear regression. Data are initially arranged in a table with two columns, the first indicating the amount of culture applied to the test (on the basis of the dilution), the other the percentage of specific lysis (see the two far-right columns of Table3).
The amount of culture that gives a lysis of 30% can thus be determined by applying a four-parameter logistic regression, or another of the proposed models, that better fit the experimental results, obtaining a measurement of L.U.30. Normalize this number on a per culture basis. To get the number of L.U.30 contained in the culture:
no. of L.U.30=
1 L.U.30
Normalize the test culture against the control culture without myeloid suppressor cells: % L.U.30=
no. of L.U.exper30 no. of L.U.ctrl30
This will give a measure of the percentage of L.U.30 in test culture compared to control culture and represent a direct measure of how much inhibition is provided by the presence
Table 3 Example of the Determination of the Culture Dilution Necessary to Obtain a Given Lysis Percentagea
Dilution number Amount of culture Percent lysis
1st 1.00E+00 97 2nd 3.33E-01 83 3rd 1.11E-01 80 4th 3.70E-02 60 5th 1.23E-02 31 6th 4.12E-03 15 7th 1.37E-03 7 8th 4.57E-04 4
aRegarding the amount of culture (see equations in Support Protocol), we assume that the initial undiluted culture is 1
and that the second well, i.e., 1:3 of the initial culture, will be 0.33 and so on. If the reference culture, without suppressive cells, produces 30% lysis of target cells at the fifth dilution, 1 liter.U.30 of the culture will be 0.0123 (1/81) of the initial culture and the number of lytic units per culture will be 1/0.0123= 81. At the same time, if a culture containing suppressive cells produces 30% lysis of target cells at the third dilution, 1 liter.U.30of this experimental culture will be 0.11 (1/9) of the initial culture and the number of lytic units per culture will be 1/0.11= 9. The % L.U.30of the experimental culture in relation to the reference culture will therefore be 9/81× 100 = 11%.
also be reported as absolute number of cells by counting the cells in the wells of culture plates before dilution.
BASIC PROTOCOL 6
MEASURING MYELOID CELL–DEPENDENT IN VIVO TOLERANCE BY ADOPTIVE TRANSFER
Immune suppression can also be studied in vivo, and the method is certainly more challenging than the in vitro setup. We previously presented a method (Marigo et al., 2010; also see previous version of this unit; doi: 10.1002/0471142735.im1417s91) to evaluate in vivo tolerance, by which normal healthy recipients were antigen stimulated and where MDSCs were adoptively transferred. This protocol permits the evaluation of tolerance in a situation of stimulation relatively free from the multiple variables present during either tumor growth or infectious diseases. Instead, the protocol reported below is extended to tumor-bearing mice where MDSCs are expanded in vivo, during tumor development, and the antigen is expressed by the tumor and is cross-presented to lymphocytes. The experiment must take into account at least three groups of animals: a group of free mice, one of tumor bearing mice and a group of vaccinated tumor-bearing mice (Ugel et al., 2012).
Materials
Dendritic cell (DC) preparation started 5 days before beginning this protocol (Inaba, Swiggard, Steinman, Romani, Schuler, & Brinster, 2009)
EG7-OVA cells (ATCC no. CRL-2113TM) stably expressing OVA257-264
Serum-free RPMI 1640 medium (e.g., Invitrogen)
CD45.2+C57BL/6 mice purchased from Jackson Laboratories RPMI containing 3% FBS (see recipe)
CD45.1+transgenic OT-1 mice obtained in our animal facility by crossing the two strains purchased from Jackson Laboratories
CD8α+T cell isolation kit (Miltenyi Biotech) RPMI containing 10% FBS (see recipe)
1 mg/ml LPS stock solution (Sigma, cat. no. L-4516)
10 mg/ml OVA257-264 peptide stock solution (available lyophilized from JPT
Peptide Technologies) Solito et al.
Dulbecco’s phosphate-buffered saline (DPBS; Lonza BioWhittaker, cat. no. BE17-515Q)
BD Golgi Stop (BD Biosciences)
FcR blocking reagent (clone 2.4G2, ThermoFisher)
Anti–mouse CD8 (i.e., PeCy5-anti CD8a, clone 53.6.7; ThermoFisher, Catalog # 15-0081-81)
Anti–mouse CD451.1 conjugated to a brilliant fluorochrome (i.e., PE-anti CD45.1, clone A20; ThermoFisher, Catalog # 12-0453-81)
BD Cytofix/Cytoperm kit (BD Biosciences)
Anti–mouse IFN-γ (i.e., FITC-anti IFN-γ, clone XMG1.2; ThermoFisher, Catalog # 11-7311-41) or rat IgG1 as isotype control (ThermoFisher)
50-ml conical tubes 1-ml syringe 18-G and 26-G needles 10-mm culture dish 2-ml syringe Refrigerated centrifuge Infrared heat lamp Mouse restraining device
100-μm nylon-mesh cell strainer (BD Biosciences) 12-ml and 4-ml round-bottom tubes
48-well culture plate
96 well U-bottom microtiter plate Microtiter plate carrier for centrifuge LSRII flow cytometer (BD Biosciences) FlowJo 7.6.5 Software (TreeStar) Red cell ACK lysis buffer (Lonza)
Additional reagents and equipment for culturing dendritic cells Inaba et al., 2009), harvesting spleens and other lymphoid organs from mice (Reeves & Reeves, 1992), counting viable cells by trypan blue exclusion (Strober, 2001), flow cytometry, injection of mice, to prepare immunosuppressive cells (Basic Protocol 1 or 2), euthanasia of mice (Reeves & Reeves, 1992), and flow cytometric IFN-γ intracellular staining
NOTE: 5 days before starting, set up a dendritic cell (DC) culture from mouse bone
marrow as described in Inaba et al. (2009), which will be used to vaccinate some groups of control mice.
Day 0
1. Collect EG7-OVA suspension cell line expressing OVA257-264from 75-cm2flasks in
a 50-ml conical tube.
2. Gently pipet cells up and down with a 1-ml syringe and an 18-G needle. 3. Load the syringe and tap bubbles, change the needle to a 26-G size.
4. Dilute an aliquot of suspension in trypan blue solution, and determine viable cell concentration (Strober, 2001).
5. Adjust EG7-OVA cell concentration to 10 × 106 cells/ml with serum-free RPMI and inject 100µl (106EG7 cells) subcutaneously into the flank of at least 5 mice
per group of CD45.2+ C57BL/6 mice.
Also prepare uninjected controls.
Day 7
6. Collect spleens (Reeves & Reeves, 1992) from OT-1CD45.1+ mice in a small volume of RPMI containing 3% FBS under sterile conditions.
7. Place spleens in a 10-mm culture dish with a small volume of RPMI containing 3% FBS and gently disaggregate using the plunger of a 2-ml syringe.
8. Add 5 ml of RPMI containing 3% FBS to cells and collect the cells in a 50-ml conical tube by gently pipetting. Repeat until the spleen pulp is completely disaggregated. 9. Centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant. 10. Dilute an aliquot of suspension in trypan blue solution, and determine viable cell
concentration (Strober, 2001).
11. Prepare CD8+T cells by means of CD8α+T cell isolation kit from Miltenyi Biotech.
Alternatively, the whole spleen of the OT-1 CD41.1+mouse could be used without any sorting. Determine abundance of CD8+T cells by means of cytometric staining with anti-CD8 and anti-Vα2 Vβ5.1/5.2, and collect an amount of total splenocytes that corresponds to 5× 106CD8+/V
α2 Vβ5.1/5.2 cells.
12. Wash enriched CD8+ T cells with at least 10 ml fresh serum-free RPMI 1640 medium twice, each time by centrifuging cells 6 min at 300×g, 4°C, and carefully discarding the supernatant.
13. Resuspend cells in a small volume of serum-free RPMI.
14. Dilute an aliquot of suspension in trypan blue solution, and determine viable cell concentration (Strober, 2001).
15. Adjust concentration to 25× 106 cells/ml with serum-free RPMI, place cells in a 50-ml conical tube, and put the tube on ice.
16. Warm CD45.2+C57BL/6 mice under an infrared heat lamp for about 10 min.
Do not overheat mice, and do not exceed the correct number of mice per cage during warming steps.
17. Gently pipet cells up and down with a 1-ml syringe and a 18-G needle. 18. Load the syringe, change the needle to a 26-G size, and tap bubbles out.
Take care to eliminate bubbles thoroughly; otherwise, mice might die from air embolism.
19. Place the mouse in the restraining device and inject 200μl of cell suspension (5 × 106cells/mouse in the tail vein).
For randomization purposes, label mice before cell injection and keep them together in the same cage.
Day 8
DC maturation
20. Gently remove supernatant medium from the DC culture. Take care to avoid scraping the plate while removing the supernatant.
21. Feed cells with 1 ml of 10RPMI/10% FBS medium containing 1μg/ml LPS (add from 1 mg/ml stock) and 2μg/ml OVA257-264peptide (add from 10 mg/ml stock).
22. Incubate the cell culture at 37°C, in a 5% CO2incubator.
Day 9
DC preparation
23. Gently remove cells from plate culture, washing wells several times with DPBS, and transfer cell suspension to a 50-ml conical tube.
24. Centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant. 25. Wash cell pellet by adding 10 ml serum-free RPMI; repeat steps 19 and 20 twice. 26. Resuspend mature DC preparation in a small volume of serum-free RPMI.
27. Dilute an aliquot of suspension in trypan blue solution, and determine viable cell concentration (Strober, 2001).
28. Adjust concentration to 10× 106 cells/ml with serum-free RPMI, place cells in a 50-ml conical tube, and put them on ice.
29. Check mature DC preparation by flow cytometry for CD11c expression and MHC II and CD80/CD86 up-regulation relative to immature DCs, as described in Inaba et al., (2009).
Perform DC vaccination
This procedure is used as a control to make sure that the in vivo tolerance is maintained in the presence of further stimulation of CD8+T lymphocytes.
30. Gently pipet mature DCs up and down with a 1-ml syringe and 18-G needle. 31. Load the syringe, change the needle to a 26-G size, and tap to remove bubbles. 32. Inoculate 100μl of DC suspension subcutaneously into each mouse flank.
Day 14
Lymph node cell preparation
33. Euthanize mice and collect inguinal, axillary, and brachial lymph nodes (Reeves & Reeves, 1992) from both sides in a small volume of RPMI/3% FBS in a 48-well plate, separately for each mouse.
34. Put lymph nodes on a nylon mesh and disaggregate mechanically with the plunger from a 2-ml syringe.
35. Let disaggregated cells pass through the nylon mesh, washing with 5 ml of RPMI containing 3% FBS. Repeat steps 34 and 35 for every lymph node until complete disaggregation is achieved.
36. Centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant.
Aspiration of supernatant with a glass Pasteur pipet attached to a vacuum pump minimizes cell loss.
37. Add 10 ml of RPMI/10% FBS to wash pellet.
38. Centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant. 39. Resuspend cell pellet in a small volume of RPMI/10% FBS and filter through a
nylon mesh into a 12-ml round-bottom tube.
40. Dilute an aliquot of suspension in trypan blue solution, and determine viable cell concentration (Strober, 2001).
41. Adjust concentration to 5× 106cells/ml with RPMI/10% FBS and place cells on
Lymph node cell staining
42. Plate lymph node cell suspension in 100μl of RPMI/10% FBS per well of a 96-well U-bottom microtiter plate.
Usually, 12 replicates, subdivided into 6 wells for specific-peptide stimulation and 6 wells for either unstimulated or unrelated peptide-stimulated control culture, should be sufficient.
43. Add 100μl of RPMI/10% FBS containing 2 μg/ml OVA257-264 peptide (add from
10 mg/ml stock) to half of the wells and 100μl of RPMI/10% FBS without peptide (or unrelated peptide) to the other half.
IL-2 could be also added, at a final concentration of 20 U/ml, to each well to increase the signal in subsequent flow cytometric analysis.
44. Incubate plate at 37°C, in a 5% CO2incubator 6 to 8 hr before Golgi stop treatment.
Ex vivo peptide stimulation must be carried out for at least 18 hr, and BD Golgi Stop treatment should not exceed 8 to 12 hr. It may be necessary to initially test and coordinate these concomitant steps in the initial setup experiments.
BD Golgi stop incubation
45. Prepare sufficient 11× BD Golgi Stop stock solution according to the manufac-turer’s instructions in RPMI/10% FBS and distribute among the wells of the plate at 20μl/well.
The BD Golgi Stop datasheet suggests using 4μl for 6 ml (final volume); hence, to fill an entire plate, prepare 1.986 ml of RPMI/10% FBS medium and add 14.7μl BD Golgi Stop.
46. Incubate 8 to 12 hr at 37°C, in a 5% CO2incubator.
Day 15
47. Transfer the cells to new 4-ml round-bottom tubes.
48. Wash samples once in DPBS, centrifuge the suspension 6 min at 300×g, 4°C, and discard the supernatant.
49. Resuspend cell pellets in 200 μl DPBS and plate 100 μl of cell suspension in duplicate on a new 96-well U-bottom microtiter plate.
It is necessary to split every sample into two aliquots in order to stain them with IFNγ -specific antibody and matched isotype control. Remember to save a small aliquot of each sample and pool together. These extra samples will be used to perform single staining controls.
50. Centrifuge the plate in a microtiter plate carrier 2 min at 300×g, 4°C, and discard the supernatant by decanting.
51. Incubate the cells with 2μg of FcR blocking reagent (2.4G2 mAb), in a volume of 50μl per well of DPBS for 20 min at room temperature.
It may be necessary to titrate the antibody to determine the appropriate amount.
52. Stain cells with 50 μl DPBS per well containing anti-CD8-PeCy5 (0.5 μl) and anti-CD45.1-PE (0.1μl) for 20 min at 4°C.
Remember to include appropriate single-staining controls. It may be necessary to titrate the antibodies.
53. Wash samples once with 100 μl/well DPBS, centrifuge the suspension 6 min at 300 ×g, 4°C, and discard the supernatant. Repeat washing with 200 μl DPBS, centrifuging under the same conditions.
54. Add 100μl/sample Fixation/Permeabilization solution (from BD Cytofix/Cytoperm kit), and gently resuspend with a multichannel pipettor. Incubate 20 min at 4°C.
55. Wash samples once with 100 μl of 1× BD Perm/Wash buffer (from BD
Cytofix/Cytoperm kit), centrifuge the plate 2 min at 300 ×g, 4°C, and discard the supernatant. Repeat washing with 100 μl of 1× BD Perm/Wash buffer and decant the supernatants.
56. Stain cells with 50μl BD Perm/Wash buffer containing in our case rat anti–mouse IFN-γ FITC (0.75 μl/well) or rat IgG1 FITC isotype control (0.75 μl/well) for 30 min at 4°C.
Remember to include appropriate single staining controls. It may be necessary to titrate antibody amount.
57. Wash samples once with 100 μl 1× BD Perm/Wash buffer, centrifuge the plate 2 min at 300×g, 4°C, and discard the supernatant by decanting. Repeat washing with 100μl 1× BD Perm/Wash buffer, and centrifuge again as before.
In order to detect intracellular IFN-γ , gate cells first by their morphology, and then collect as many events as possible in the CD8+/CD45.1+ gate. After the acquisition, proceed with data analysis. For each sample, either stained with specific antibody or isotype control, provide the same gating schema, taking care to adapt gates for minor changes. Gate cells by morphology and identify CD8+CD45.1+populations. Inside this gate, set a baseline gate on negative IFN-γ staining using the isotype control, and copy this gate to the sample stained with the specific antibody, in order to determine the percentage of IFN–γ -releasing lymphocytes.
BASIC PROTOCOL 7
IDENTIFICATION AND SORTING OF HUMAN M-MDSC CELLS FROM PERIPHERAL BLOOD (PB) OF CANCER PATIENTS TO MEASURE THEIR IMMUNOSUPPRESSIVE ACTIVITY
The aim of this protocol is to obtain CD14+ cells enriched for M-MDSCs from PB of cancer patients, and to preserve their functional activity in order to use them for in vitro functional assays. PBMCs are obtained after centrifugation and sedimentation of whole blood on a Ficoll gradient (see Mandruzzato et al., 2009).
The selection of fluorochrome-labeled antibodies to identify M-MDSCs has to be per-formed based on optimal signal strength and minimal spectral overlap; the optimal concentration of each antibody must be evaluated in titration experiments.
Materials
Anticoagulated whole blood obtained from cancer patients Sorting buffer (see recipe)
Human FcR blocking reagent (Miltenyi Biotec) CD14 FITC (clone M5E2, BD Biosciences) HLA-DR APC (clone L243, BD Biosciences) Complete IMDM medium (see recipe) 15- and 50-ml polypropylene conical tubes Refrigerated centrifuge
12× 75–mm polypropylene tubes pre-coated for at least 1 hr with heat-inactivated fetal bovine serum (FBS)
0 50K 100K 150K 200K 250K FSC-A 0 50K 100K 150K 200K 250K SSC-A SSC-A 0 102 103 104 105 HLA-DR 0 102 103 104 105 CD14 CD14 doublets exclusion MIX FMO Morphological gate on PBMC Gate on monocytes 0 102 103 104 105 CD14 0 102 103 104 105 FMO 0 50K 100K 150K 200K 250K 0 102 103 104 105 CD14 CD124 CD14 FM O 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 Gate on CD14 /HLA-DR+ lowcells
Gate on CD14 /CD124 cells+ +
Figure 3 Gating strategy for the definition of M-MDSCs in human PBMCs. PBMCs were labeled with anti-CD14, anti-HLA-DR, and anti-CD124, and then analyzed by flow cytometry. After a morphological gate on PBMCs and doublets exclusion, monocytes were gated as CD14+cells, and then the gate for HLA-DRlowcells and CD124+ cells was set based on the FMO control.
Additional reagents and equipment for isolating PBMC from whole blood (Mandruzzato et al., 2009) and counting viable cells (Strober, 2001)
NOTE: All the procedures, including sorting, are performed at 4°C, to avoid cell loss and adherence to the plastic.
Determination of M-MDSCs expression level on PBMCs
The following antibodies were used to characterize blood M-MDSCs in melanoma (Damuzzo et al., 2016) and meningioma (Pinton et al., 2018) patients: CD14-FITC, HLA-DR-APC and CD124 PE. As the down-regulation of HLA-DR and CD124 expression are important parameters to determine two subsets of M-MDSCs, it is important to use FMO controls to define HLA-DR and CD124 gates as shown in Figure 3. The protocol for PB-M MDSC staining is described below:
1. Isolate PBMCs from whole blood of cancer patients (see Mandruzzato et al., 2009). Collect cells in 12× 75–mm tubes for FACS analysis and wash them with sorting buffer. Centrifuge 6 min at 300× g, 4°C, and discard the supernatant.
2. After the centrifugation, tubes are subsequently incubated with Fc-receptor blocking reagent diluted 1/25 at 4°C for 10 min.
3. Add the mixture of antibodies (CD14/HLA-DR/CD124) to the tubes in a final volume of 100μl sorting buffer and incubate at 4°C for 20 min.
Common mAbs used for characterization of human CD11blow/- BM-MDSCs and PB
M-MDSCs are listed in Table 2.
4. At the end of the incubation time, wash cells with sorting buffer. Centrifuge the suspension for 6 min at 300× g, 4°C, and discard the supernatant.
5. Resuspend samples in 250μl of sorting buffer, and proceed with flow cytometric acquisition and analysis.
